Linking agents and methods

ABSTRACT

Compounds containing carbohydrate moieties or carboxyl groups are linked to either compounds containing thiol moieties or electron-deficient moieties by the use of linking agents of the formula ##STR1## in which R 1  is NH 2  -- or NH 2  --NH--; R 2  is --NH--C(O)--, --C(O)--NH--, or --C(O)--; R 3  is C 1  -C 10  alkylene, C 5  -C 7  cyclic alkylene, arylene, phenyl-substituted C 1  -C 10  alkylene, benzyl-substituted C 1  -C 10  alkylene, or amino-substituted C 1  -C 10  alkylene; R 4  is H, acetyl, ##STR2## where R 5  is C 1  -C 5  alkyl; m is zero or 1; and n ia zero or 1.

This is a continuation of application Ser. No. 07/151,700, filed Feb. 3,1988, now U.S. Pat. No. 4,970,303.

BACKGROUND OF THE INVENTION

This invention relates to the formation of conjugates, and in particularto the joining of one species to another at a carbohydrate or carboxylmoiety on one of the species, utilizing a thioether or disulfide bond aspart of the linkage.

The linkage of compounds of various types to carbohydrate or carboxylmoieties is desirable for a variety of reasons. In the formation ofconjugates involving immunoglobulins, for instance, linkage at specificregions on the immunoglobulin is often desirable for purposes ofmaintaining the accessibility of antigen-binding sites or of sites onthe Fc chains for complement binding. As a further example, certainsolid supports used in affinity chromatography have carbohydrate orcarboxyl groups available for binding, and the same is true for othersolid phase materials as well such as those used in two-phaseimmunoassays.

Many of the species which are sought to be linked to these carbohydrateor carboxyl moieties are species which lack the ability to reactdirectly.

SUMMARY OF THE INVENTION

Novel compositions and methods are provided herein for the formation oflinkages of the type described above at carbohydrate or carboxylmoieties. The novel compositions are linking agents falling within thefollowing generic formula: ##STR3## in which R¹ is a member selectedfrom the group consisting of NH₂ and NH₂ --NH--;

R² is a member selected from the group consisting of --NH--C(O)--,--C(O)--NH--, and --C(O)--;

R³ is a member selected from the group consisting of C₁ -C₁₀ alkylene,phenylsubstituted C₁ -C₁₀ alkylene, benzylsubstituted C₁ -C₁₀ alkylene,aminosubstituted C₁ -C₁₀ alkylene, C₅ -C₇ cyclic alkylene and arylene;and

R⁴ is a member selected from the group consisting of H, acetyl, ##STR4##where R⁵ is C₁ -C₅ alkyl: m is zero or 1; and

n is zero or 1.

Within the scope of this formula, certain embodiments are preferred,notably those of the formulas given below. (In each of these formulas,R¹, R³, and R⁴ are as defined above.) ##STR5##

In each of these formulas, the terms "alkylene" and "alkyl" refer tosaturated divalent and monovalent hydrocarbon radicals, respectively,and are intended to include straight-chain, branched-chain and cyclicstructures. Examples of alkylene groups are --CH₂ --, --CH₂ --CH₂ --,--CH₂ --CH₂ --CH₂ -- and longer chains; and --CH(CH₃)--, --C(CH₃)₂ --,--CH(CH₃)--CH₂ --, --CH(CH₃)--CH(CH₃)--, --CH(CH₃)₂ --CH₂ --, ##STR6##and the reverses thereof (i.e., left-to-right). Preferred alkylenegroups are those having 1 to 6 carbon atoms, the most preferred beingthose having 1 to 4 carbon atoms.

The term "cyclic alkylene" refers to a saturated divalent cyclichydrocarbon radical, with the two points of attachment being at any twolocations on the ring. Examples are cyclopentylene, cyclohexylene, andcycloheptylene. A preferred group is 1,2-cyclohexylene (in which the twopoints of attachment are on adjacent carbon atoms on the ring).

The term "arylene" refers to a divalent radical containing at least onearomatic ring, with the two points of attachment being at any twolocations on the ring (or rings, in the case of multi-ring groups).Examples are phenylene, particularly 1,2-phenylene, and naphthylene.

In Formulas I, II, III and IV, the two points of attachment on thephenyl ring may be ortho-, meta- or para- with respect to each other.Compounds in which the points of attachment are meta- or para- arepreferred, with para- the most preferred. In the R⁴ definition, thesulfur substituent on the pyridine and pyridine N-oxide may be in the2-, 3-, or 4-position. The 2- and 4-positions are preferred, with the2-position being the most preferred.

The following are examples of compounds within these formulas:

S-acetyl-4-(4-aminophenyl)-1-butanethiol

S-acetylthioacetic acid, 4-aminoanilide

2-pyridyl-3'-propanoyl disulfide, 4-aminoanilide

S-acetylthioacetic acid hydrazide

2-pyridyl-3'-propanoyl disulfide hydrazide

2-pyridyl-1'-methyl-3'-propanoyl disulfide hydrazide

2-pyridyl-1',1'-dimethyl-3'-propanoyl disulfide hydrazide

2-pyridyl-1',2'-dimethyl-3'-propanoyl disulfide hydrazide

2-pyridyl-2',2'-dimethyl-3'-propanoyl disulfide hydrazide

2-pyridyl 1'-(2'-ethanoyl)-cyclopropane disulfide hydrazide

2-pyridyl-1',1'-dimethyl-2'-amino-3'-propanoyl disulfide hydrazide

2-pyridyl-1'-isopropyl-2'-ethanoyl disulfide hydrazide

2-pyridyl-1'-phenyl-2'-ethanoyl disulfide hydrazide

2-pyridyl-1'-methyl-2'-ethanoyl disulfide hydrazide

2-pyridyl-1',1'-dimethyl-2'-ethanoyl disulfide hydrazide

2-pyridyl 1'-(2'-ethanoyl)-cyclohexane disulfite hydrazide

2-pyridyl 1'-(2'-ethanoyl)-cycloheptane disulfide hydrazide

The invention further extends to water-soluble salts and derivatives ofthese compounds. The salts may be formed by anionic moieties tocomplement the amine (or hydrazine) terminus of the compounds incationic form. Examples of such salts are acetate salts,trifluoroacetate salts, hydrohalide salts, particularly hydrochlorideand hydrobromide salts, and toluenesulfonic acid salts. The derivativesmay be compounds having the same formula but with the addition of acharge group at a point on the molecule where it does not interfere withthe coupling ability of either of the end groups on the molecule.Examples of such derivatives are those bearing sulfonic acid (--SO₃ --)groups and those bearing nonreactive amino groups (such as diethylamino,for example). The most preferred among these are the trifluoroacetatesalts.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The compounds of the present invention are prepared by conventionaltechniques well known among those skilled in the art, selected inaccordance with the desired substituent groups of the formulas as listedabove. Compounds with aminophenyl terminal groups (as in Formulas II,III and IV with R¹ =NH₂ --), for instance, may be prepared from thecorresponding nitrophenyl analogs by reduction. An example is reductionwith stannous chloride. Compounds where R¹ is a hydrazino group may beprepared from an N-protected hydrazino starting material or anappropriately substituted carbazate.

Compounds of Formula II may be prepared from the correspondingnitrophenyl alcohols. Compounds of Formula III with an amino group as R¹may be prepared by reacting an appropriately substitutedN-hydroxysuccinimide ester with an appropriately substituted4-aminoaniline. Other standard coupling methods for this formula mayalso be used.

Compounds with a carbonylaminomethylene group may be prepared bycoupling an N-protected amino benzoic acid with the appropriate aminodisulfide or S-protected amino thiol. Compounds of Formula IV with ahydrazino group as R¹ may be prepared by coupling an N-protectedhydrazino benzoic acid with the appropriate thiol-containing amine.Compounds of Formula V with a hydrazino group as R¹ may be prepared bycoupling a thiol-containing carboxylic acid with an appropriatelysubstituted carbazate.

The preparation of other compounds within these formulas, as well as thederivatives described above, is done with the appropriate variations onthe above, as will be readily apparent to those skilled in the art.

Salts are readily prepared by conversion of the compounds in carbamateform with the appropriate acids, and the free base may be formed bytreatment with base.

These compounds are useful in placing a sulfhydryl or disulfidefunctional group at the site of a carbohydrate group or a carboxylmoiety on another compound, and likewise for linking two compounds atspecified functional groups, i.e., a carbohydrate or carboxyl moiety onone and a thiol or electron-deficient moiety on the other. In the caseof the thiol moiety, the reaction at the corresponding end of thelinking agent will produce a disulfide group, preferably by disulfideexchange. In the case of an electrondeficient moiety (such as maleimideor an α-halo carbonyl group), the reaction will be a nucleophilicdisplacement or addition reaction.

The utility of these linking agents is in providing linkages betweenspecies in a site-specific manner with respect to at least one of thespecies, and in some cases with site specificity on both species. Theseagents may thus be used for example in linking proteins or othermolecules to carbohydrates or carboxyl groups on other proteins orsubstances such as column supports and glass, or any substancecontaining the particular types of functional groups with whichphenylamines, hydrazines or hydrazides will react. They can also be usedto introduce a sulfhydryl moiety for purposes of site specificity forreaction with an electron-deficient moiety of another species, such asfor example an ethylene group between two highly electronegative groups(i.e., maleimide).

The linking reactions used to form these linkages may be done accordingto conventional techniques well known among those skilled in the art. Inthe case of a species containing a carbohydrate moiety, the carbohydrateis first converted to an aldehyde. It is then reacted with the aminoterminus of the linking agent in accordance with known conventionalreaction conditions to form an imine or hydrazone linkage which can thenbe reduced if necessary. When more than one carbohydrate moiety ispresent on the species, certain specific carbohydrates may beselectively oxidized in some cases, depending on the type of moleculebearing the carbohydrate groups. In the case of immunoglobulins, forinstance, carbohydrate side chains may be selectively oxidized by theuse of galactose oxidase or periodate under mild conditions. Thereaction at the other end of the linking agent, as mentioned above, willdepend on whether the reaction is one of disulfide exchange ornucleophilic displacement. In either case, conventional procedures knownto those skilled in the art may be used.

In the case of a species containing the carboxyl moiety the aminoterminus of the linking agent is coupled to form an amide bond.

A wide variety of pairs of species may be linked by the linking agentsof the present invention. Examples are proteins and macromolecules ingeneral, linked to other proteins or macromolecules or to smallermolecular species, such as bifunctional chelators, luminescent agentsand NMR shift reagents. A particularly useful example is the coupling ofimmunoglobulins at the Fc region to toxins or labels to impart the sitespecificity characteristic of the immunoglobulin to the toxin or label.Examples of such labels are enzymes, radioisotopes (through bifunctionalchelators), and fluorescent agents (also possibly through bifunctionalchelators).

The following examples are offered for purposes of illustration, and areintended neither to limit nor define the invention in any manner. Inthese examples, the following abbreviations are used:

NMR: nuclear magnetic resonance at 60 MHz; all chemical shifts given inδ values relative to tetramethylsilane: "s"=singlet, "br s"=broadsinglet, "d"=doublet, "t"=triplet,

"m"=multiplet, "ar."=aromatic

IR: infrared spectra; values given in cm⁻¹ ; "sh" =shoulder, "br"=broad

LRMS: low resolution mass spectroscopy, intensity given relative to thebase peak

TLC: thin-layer chromatography: values given in R_(f) (ratio to thefront)

UV/VIS: ultraviolet/visible spectra: values given in relative absorption

TFA: trifluoroacetic acid

EtOAc: ethyl acetate

Ac: acetyl

s.m.: starting material

EXAMPLE 1 Preparation of S-Acetyl-4-(4-aminophenyl)-1-butanethiol

This example illustrates the preparation of one of the compounds withinthe scope of the present invention.S-acetyl-4-(4-aminophenyl)-1-butanethiol, whose structural formula isthat of Formula I above, in which R¹ is 4-amino, R³ is --(CH₂)4-- and R⁴is acetyl.

A solution of S-acetyl-4-(4-nitrophenyl)-1-butanethiol (1.0 g. 3.95mMol) in 25 mL of methanol was heated in a bath at 60° C. under anitrogen atmosphere with SnCl₂.2H₂ O (4.45 g, 19.8 mMol) for 6 hours.The composition was not changing after 4 hours (using thin-layerchromatography, hereinafter "TLC"), although some starting materialstill remained. (The product was observed to have a R_(f) of 0.0 in a90:10 mixture of hexanes and ethyl acetate, as opposed to a R_(f) of0.45 for the starting material.) An additional 0.60 g of SnCl₂.2H₂ O wasthen added. After 6 hours, the reaction mixture was cooled on ice andthen brought to pH 7 with 100 mL of cold 50% saturated aqueous NaHCO₃.

A voluminous white precipitate formed. This was extracted with four 100mL portions of ethyl acetate. The organic layers were rinsed with water,dried over Na₂ SO₄, and concentrated in vacuo. NMR indicated thepresence of the thioacetyl group (a positive indication of the existenceof the desired product). The crude yield was 0.60 g (68%).

The crude product was subjected to flash chromatography on SiO₂, elutingwith 90/10 hexanes/ethyl acetate to 80/20 hexanes/ethyl acetate. Themajor product eluted with 20% ethyl acetate and had a R_(f) of 0.08 in90/10 hexanes/ethyl acetate, yielding 300 mg of an oil (34% yield).Identity of the product as S-acetyl-4-(4-aminophenyl)-1-butanethiol wasconfirmed by NMR, TLC, IR, LRMS and UV/VIS as follows:

NMR (CDCl₃); 6.88 (d. 2H, ar.); 6.50 (d. 2H, ar.); 3.48 (s, 2H, NH₂)2.85 (m, 2H, CH₂); 2.47 (m, 2H, CH₂); 2.32 (s, 3H, SAc); 1.57 (m, 4H,2CH₂)

IR (NaCl, neat); 3440, 3390 (NH₂); 2930, 1680 (s, SAc); 1618, 1528(NH₂); 1278, 1135, 960, 830 (strong NO₂ absorbance at 1350 cm⁻¹ in s. m.gone)

LRMS: 223 (M⁺, 14.0%); 181 (M⁺, --COCH₃, 22.0%); 106 (100%)

TLC (90/10 hexanes/EtOAc); R_(f) 0.08 (ninhydrin positive)

UV/VIS: (CHCl₂) 296 (0.33), 250 (1.41); (CH₃ OH) 288 (0.05), 234 (0.69),214 (0.85)

EXAMPLE 2 Preparation of 4-(4-Aminophenyl)-1-butanethiol

The structural formula of this compound corresponds to that of Formula Iabove, in which R¹ is 4-amino, R³ is --(CH₂)4-- and R⁴ is H.

A solution of S-acetyl-4-(4-aminophenyl)-1-butanethiol (1.3 g. 5.8 mMol)in ethanol (25 mL) was stirred with 1M aqueous ammonia (25 mL) at roomtemperature in the dark under nitrogen overnight. The pH was thenadjusted to 6.0 with 2M HCl and the solution extracted with three 75-mLportions of EtOAc. The organic layers were rinsed with water dried overNa₂ SO₄ and concentrated in vacuo to give a pale yellow liquid weighing0.94 g (90% yield). The identity of the product as4-(4-aminophenyl)-1-butanethiol was confirmed by NMR, TLC, IR and UV/VISas follows:

NMR (CDCl₃); 6.92 (d, 2H, ar.); 6.53 (d. 2H, ar.); 3.53 (s, 2H, NH₂);2.28-2.75 (m, 4H); 1.08-1.92 (m, 5H)

IR (NaCl, neat); 3440, 3360, 3020, 2930, 2860, 1620 (s), 1515 (s), 1435,1280 (s), 1185, 1130, 830

TLC (50/50 hexanes/EtOAc): R_(f) 0.65

UV/VIS: (CH₂ Cl₂) 296 (0.261), 246 (1.05); (CH₃ OH) 290 (0.147), 238(1.02), 210 (0.702)

EXAMPLE 3 Preparation of 2-Pyridyl-4'-[1-(4-aminophenyl)]butyl Disulfide

The structural formula corresponds to that of Formula I above, in whichR¹ is 4-amino, R³ is --(CH₂)₄ -- and R⁴ is 2-pyridylthio. A solution of4-(4-aminophenyl)-1-butanethiol (0.94 g, 5.2 mMol) in EtOAc (20 mL) wastreated with 2,2'-dipyridyldisulfide (1.15 g. 5.2 mMol) as a solid.After all the solid had dissolved, 4 drops of BF₃.(C₂ H₅)₂ O was addedand the solution was heated at a bath temperature of 55° C. Thedisulfide exchange was sluggish, so after 2 hours, 17 mL of benzene wasadded and the temperature was increased to 65° C. After a further 20hours, the solvent was removed in vacuo and the crude product waspurified on a chromatotron (SiO₂, Harrison Research), eluting with astep gradient of 5-20% EtOAc in hexanes. The mixed disulfide wasobtained as a pale yellow oil weighing 0.41 g (27% yield). The identityof the product as 2-pyridyl-4'-[1-(4-aminophenyl)]butyl disulfide wasconfirmed by NMR. TLC and IR as follows:

NMR (CDCl₃); 8.45 (m. 1H, pyridyl); 7.42-7.82 (m, 2H, pyridyl);6.75-7.18 (m, 1H, pyridyl); 6.92 (d, 2H, phenyl); 6.55 (d, 2H, phenyl);3.58 (s, 2H, NH₂); 2.25-3.00 (m, 4H, 2CH₂); 1.38-2.00 (m, 4H, 2CH₂)

IR (NaCl, neat) 3430, 3340, 2930 (s), 1620, 1575, 1520, 1450, 1420,1280, 1120, 830, 765

TLC (50/50 EtOAc/hexanes): R_(f) 0.74

EXAMPLE 4 1. Preparation of S-Acetylthioacetic Acid.4-(t-Butoxycarbonylamino)anilide

This compound was prepared as a precursor to the trifluoroacetate salt(described below).

A solution was prepared consisting of S-acetylthioacetic acid.N-hydroxysuccinimide ester (1.29 g. 5.6 mMol) and4-(t-butoxycarbonylamino)aniline (1.5 g. 5.6 mMol) in ethyl acetate (30mL). The solution was stirred at room temperature for 3.5 hours, then at45° C. overnight. The reaction mixture remained colorless throughout.

The reaction mixture was then rinsed with two 50 mL portions of 10%saturated aqueous NaHCO₃, then with 50 mL of H₂ O. The organic layerswere dried over Na₂ SO₄ and concentrated in vacuo to give a yellow oil.This was flash chromatographed twice on a SiO₂ column slurried in 50/50ethyl acetate/petroleum ether, eluting with the latter as well. Thefastest running fractions from each were pooled and concentrated. Theresulting solid was recrystallized from methylene chloride/petroleumether to give 1.22 g (67% yield) of a white solid with melting point161°-162° C. The latter was identified as S-acetylthioacetic acid.4-(t-butoxycarbonylamino)anilide by IR. NMR. UV/visible, and TLC, asfollows:

NMR (CDCl₃): 8.00 (br s, 1H, NH); 7.33 (s, 5H, ar.); 6.55 (br s, 1H,NH); 3.65 (s, 2H, CH₂); 2.43 (s, 3H, SAc); 1,52 (s, 9H. t-butyl)

IR (KBr): 3350 (s), 1695 (s, SAc), 1660 (s, amide C═O) 1550 (s), 1410,1315, 1235, 1170, 1065, 825, 705, 630

UV/VIS: (CH₃ OH) 262 (0.597), 210 (0.518); (CH₂ Cl₂) 270 (sh, 0.750),258 (0.873)

TLC (50/50 EtOAc/hexanes); R_(f) 0.75

2. Preparation of S-Acetylthioacetic Acid. 4-Aminoanilide,Trifluoroacetate Salt

This compound has the structural formula of Formula III above, in whichR¹ is 4-amino. R³ is methylene, and R⁴ is acetyl, in the form of thetrifluoroacetate salt of the primary amine at the left end of thestructure as shown.

The product of Section 1 of this example in the amount of 200 mg wasdissolved in 5 mL of freshly distilled trifluoroacetic acid (hereinafter"TFA"). and stirred in the dark at room temperature under a nitrogenatmosphere for 1.5 hours. The solvent was then removed in vacuo at atemperature less than 30° C. to give a pale yellow oil in approximatelyquantitative yield. Its identity was confirmed as that ofS-acetylthioacetic acid, 4-aminoanilide. trifluoroacetate salt by NMR asfollows:

NMR (D₂ O) 7.57 (d, 2H, ar.); 7.33 (d, 2H, ar.); 3.82 (s, 2H, CH₂ SAc);2.42 (s, 3H, SAc)

EXAMPLE 5

Preparation of 2-Pyridyl-3'-propanoyl Disulfide,4-(t-Butoxycarbonylamino)anilide

This compound was prepared as the precursor of the trifluoroacetate salt(described below).

A solution of 2,2'-dipyridyl disulfide (Aldrich. 1.5 g. 6.8 mMol) in 10mL of dry ethyl acetate was treated with 3-mercaptopropionic acid (0.73g. 6.89 mMol) in 10 mL of dry ethyl acetate. Then four drops of BF3.(C₂H₅)₂ O were added and the reaction stirred overnight at room temperatureunder a nitrogen atmosphere. The reaction was concentrated to dryness invacuo, then slurried with 10 mL of cold ethyl acetate and filtered ofall solid to give a pale yellow solution. To the latter were added4-t-butoxycarbonylamino aniline (1.82 g, 6.8 mMol) in 10 mL of dry ethylacetate and dicyclohexylcarbodiimide (1.40 g, 6.8 mMol). also in 10 mLethyl acetate. The reaction was again stirred overnight at roomtemperature, then filtered to remove dicyclohexylurea. The filtrate wasconcentrated in vacuo, then subjected to flash chromatography on SiO₂,eluting with 30/70 ethyl acetate/hexanes.

A first fraction at R_(f) 0.8 (with 50/50 ethyl acetate/hexanes) was anoil not further characterized. A second fraction at R_(f) 0.62 was apale yellow solid, 0.48 g, identified by NMR as 4-t-butoxycarbonylaminoaniline. A closely following third fraction at R_(f) 0.50 was a solid,0.66g (representing 23% yield) with melting point 139°-140° C. This wasrecrystallized from ethanol to yield a solid with a melting point of154.5°-155.5° C. Its identity was confirmed as that of2-pyridyl-3'-propanoyl disulfide. 4-(t-butoxycarbonylamino)anilide byNMR, IR. and elemental analysis, as follows:

NMR (CDCl₃ /CD₃ OD): 8.30 (m. 1H, pyridyl); 7.50-7.80 (m, 2H, pyridyl);7.37 (s, 4H, phenyl ring); 6.87-7.27 (m, 1H, pyridyl); 3.00 (m, 2H);2.75 (m, 2H); 1.50 (s, 9H, t-butyl)

IR (KBr): 3340 (Br); 1690,1655 (both C═O); 1520 (s), 1390, 1165, 1075,842, 770

Elemental Analysis, found(required): C: 56.11(56.27), H: 5.83(5.72), N:10.27(10.36), S: 15.75(15.81)

2. Preparation of 2-Pyridyl-3'-propanoyl Disulfide, 4-Aminoanilide,Trifluoroacetate Salt

This compound has the structural formula of Formula III above, in whichR¹ is 4-amino, R³ is --(CH₂)₂ --, and R⁴ is 2-pyridylthio, in the formof the trifluoroacetate salt of the primary amine at the left end of thestructure as shown.

A solution was prepared consisting of 150 mg of the product of Section 1of this Example in 5 mL of freshly distilled TFA. The solution wasstirred at room temperature under a nitrogen atmosphere for 1.5 hours inthe dark. No yellow color (which would indicate liberated2-pyridylthiol) was observed. The solvent was removed in vacuo at atemperature less than 30° C. to give a solid in quantitative yield. Thesolid was soluble in water, methanol and ethyl acetate, and its identitywas confirmed as that of 2-pyridyl-3'-propanoyl disulfide,4-aminoanilide, triflueroacetate salt by proton NMR and UV/visible, asfollows:

¹ H NMR (D₂ O): 8.62 (m. 1H, pyridyl); 8.18 (m, 2H, pyridyl); 7.56-7.90(m, partially obscured, 1H, pyridyl); 7.50 (pseudo-d, 4H, phenyl ring);3.33 (t, 2H); 2.95 (t, 2H)

UV/VIS (H₂ O): 242 (with poorly defined broad absorbance to about 310nm)

EXAMPLE 6 Preparation of S-Acetylthioacetic Acid, N-t-ButoxycarbonylHydrazide

This compound has the structural formula of Formula V above in which R¹is hydrazino, R³ is methylene, and R⁴ is acetyl, in the form of thet-butyl carbamate.

A mixture of S-acetylthioacetic acid. N-hydroxysuccinimide ester (1.5g,6.49 mMol) and t-butylcarbazate (0.86 g, 6.49 mMol) was stirred in dryethyl acetate under a nitrogen atmosphere at room temperature for 24hours, then at 60° C. for an additional 6.5 hours. The reaction mixturewas then cooled on ice, rinsed with two 50 mL portions of saturatedaqueous NaHCO₃ followed by water, then dried over Na₂ SO₄ andconcentrated in vacuo. A major product spot was observed by TLC (using50/50 ethyl acetate hexanes) at R_(f) 0.42.

The crude product was subjected to flash chromatography on SiO₂ elutingwith 50/50 ethyl acetate/hexanes, which yielded a very pale yellow oil,1.22 g (76% yield). Residual starting material was crystallized out, andthe remaining mixture was concentrated to dryness, dissolved in 20 mLethyl acetate. and rinsed with four 25 mL portions of 20% saturatedaqueous NaHCO₃ followed by 25 mL of water, then dried over Na₂ SO₄ andconcentrated in vacuo to yield the final product, an oil whose identitywas confirmed as that of S-acetylthioacetic acid. N-t-butoxycarbonylhydrazide by NMR, IR, and UV/visible, as follows:

NMR (CDCl₃): 8.50 (br s, 1H, NH); 7.00 (br s, 1H, NH); 3.63 (s, 2H, CH₂SAc); 2.38 (s, 3H, SAc); 1.46 (s, 9H, t-butyl)

IR (NaCl, neat): 3290 (br), 2990, 1690 (br), 1490, 1375, 1255, 1165

UV/VIS: (CH₂ Cl₂) 248; (CH₃ OH) 218

This compound can be converted to the trifluoroacetate salt byprocedures analogous to those described in the other examples in thisspecification. The compound may also be converted to other salts withinthe scope of the present invention by other conventional procedures.

EXAMPLE 7

Preparation of 2-Pyridyl-3'-propanoyl Disulfide, N-t-ButoxycarbonylHydrazide

This compound was prepared as a precursor of the trifluoroacetate salt(described below).

A solution of 2,2'-dipyridyl disulfide (3.8 g, 17 mMol) in 20 mL ofethyl acetate was treated with 1.8 g (17.2 mMol) of 3-mercaptopropionicacid in 10 mL of ethyl acetate and 5 drops of BF₃.(C₂ H₅)₂ O. Thereaction mixture was stirred for 5 hours under nitrogen in the dark,then filtered and concentrated in vacuo. The resulting solid residue wasslurried in 20 mL of cold ethyl acetate and refiltered. Then 1.98 g (15mMol) of t-butylcarbazate was added, followed by 3.09 g (15 mMol) ofdicyclohexylcarbodiimide in 10 mL of dry ethyl acetate. The reaction wasstirred at room temperature for 18 hours in the dark, then filtered andconcentrated in vacuo to yield a yellow oil. The oil was subjected toflash chromatography on SiO₂, eluting with 50/50 ethyl acetate/petroleumether (35°-60° C.).

Three fractions were collected, the second containing the desiredproduct at R_(f) 0.40 (50/50 ethyl acetate/hexanes), 1.56 g (28% yield)of an oil, whose identity was confirmed as that of2-pyridyl-3'-propanoyl disulfide, N-t-butoxycarbonyl hydrazide by NMR,IR, and UV/visible, as follows:

NMR (CDCl₃): 9.46 (br s, 1H, NH); 8.43 (m, 1H, pyridyl); 7.63 (m, 2H,pyridyl); 7.50 (br s, 1H, NH); 7.10 (q, 1H, pyridyl); 3.07 (m, 2H, CH₂)2.77 (m, 2H, CH₂) 1.47 (s, 9H, t-butyl)

IR (NaCl, neat); 3280 (br), 2990, 1730 (sh), 1685 (br), 1420, 1372,1250, 1165, 770

UV/VIS: CH₂ Cl₂) 286 (1.05), 250 (1.30); (CH₃ OH) 284 (0.74), 238(1.49), 214 (sh. 1.11)

Preparation of 2-Pyridyl-3'-propanoyl Disulfide HydrazideTrifluoroacetate Salt

This compound has the structural formula of Formula V above in which R¹is hydrazino. R³ is --(CH₂)₂ -- and R⁴ is 2-pyridylthio, in the form ofthe trifluoroacetate salt of the primary amine at the left end of thestructure as shown.

A portion of the product of Section 1 of this Example (200 mg) wasdissolved in freshly distilled TFA (5 mL) and stirred in the dark atroom temperature under a nitrogen atmosphere for 1 hour, during whichtime a very faint pink color developed. The solvent was then removed invacuo at a temperature less than 30° C. to give a pale yellow oil inapproximately quantitative yield. The identity of the oil was confirmedas that of 2-pyridyl-3'-propanoyl disulfide hydrazide, trifluoroacetatesalt by NMR, as follows:

NMR (D₂ O); 8.73 (m, 1H, pyridyl); 8.33 (m, 2H, pyridyl), 7.88 (m, 1H,pyridyl); 3.23 (m, 2H, CH₂); 2.93 (m, 2H, CH₂)

EXAMPLE 8 Conjugate Preparation

This example illustrates the preparation of a conjugate of ricin A-chainwith IND1 antibody through a carbohydrate moiety on the latter, using alinking agent in accordance with the present invention. The linkingagent used is that prepared in Example 5, part 2. The followingabbreviations are used in this example:

RTA: Ricin toxin A-chain

NaOAc: sodium acetate

DMSO: dimethyl sulfoxide

DTT: dithiothreitol

SDS PAGE: sodium dodecyl sulfide polyacrylamide gel electrophoresis

PBS: phosphate-buffered saline

SPDP: N-succinimidyl-3-(2-pyridyldithio)propionate

EIA: enzyme immunoassay

1. Periodate Oxidation of Carbohydrate Moiety on Antibody and ReactionWith Linking Agent

1 mL of 5 mg/mL IND1 antibody in PBS was spun through a G-50 columnequilibrated with 0.1 M NaOAc+0.15M NaCl, pH 5, and diluted 1:1 with 1mL of 0.1M NaOAc buffer. The antibody was oxidized with 10 mM sodiumperiodate for 20 minutes at 0° C. in the dark, then quenched with 10 mMglycerol for 20 minutes at 0° C. in the dark. 250 μL of the quenchedreaction was spun through G-50 into fresh acetate buffer. pH 5, thendiluted 1:1 with acetate buffer to a final volume of 500 μL (3.3 μM).

A solution of the trifluoroacetate salt of 2-pyridyl-3'-propanoyldisulfide. 4-aminoanilide was prepared by combining 5-8 mg of the salt(which was a yellow oil) with 10 μL DMSO to give a 1.5-2M solution. Thiswas then diluted about 50× with ethanol to produce 30mM 10× concentratedworking stock solution. 55 μL of this solution was added to the reactionmixture described above, resulting in a final linking agentconcentration of 300 μM (a linker:antibody ratio of about 100). Thereaction was allowed to proceed for 15 h at 4° C. in the dark withgentle agitation. The solution was then treated with 10 mM NaCNBH₃(aqueous) for 4 h at 4° C. with gentle agitation. The antibody was thenspun through G-50 into SPDP buffer and concentrated to 1.3 mg/mL for thecoupling reaction with RTA.

2. Reaction With RTA and Characterization of the Immunotoxin

RTA was concentrated to 6.4 mg/mL. and treated with 50mM DTT for 1 hourat room temperature to reduce sulfhydryls, then spun through G-50 intoSPDP buffer. pH 7.5. The concentration of the reduced RTA was 4.75mg/mL. A tenfold molar excess of RTA was added to IND1 (272 μL of 4.75mg/mL RTA added to 0.5 mL 1.3 mg/mL IND1), inverted to mix and allowedto stand overnight at 4° C. without stirring. The presence ofimmunotoxin in the crude reaction was confirmed by SDS PAGE (Coomassie)and quantitated by densitometer scanning. The monoconjugate appeared tobe 36% of the crude reaction mixture. The di-conjugate was present inabout 10% yield. About 40% of the IND1 antibody was unreacted.

3. Purification of Immunotoxin

The impure reaction product was loaded on AcA-44 in SPDP. pH 7.5. Twopeaks of protein were eluted from the column; the second peak containingantibody and immunotoxin was concentrated (100 μL. 1.77 mg/mL) andloaded onto a 0.8 mL column of Affigel Blue. After loading by gravity,the column was washed with 10 volumes of PBS, pH 7 at a flow rate ofabout 15 mL/h. A high salt, high pH step was then applied (0.1Mphosphate, 0.5M NaCl, pH 8) to elute the immunotoxin. The immunotoxinwas concentrated to about 60 μL (0.9 mg/mL) for SDS PAGE and activityassays (EIA and whole cell kill).

4. Activity Assay Results

Densitometry of the Coomassie stained SDS PAGE gel showed that theAffi-gel Blue purified material contained 10% free antibody, 20%monoconjugate and 9% di-RTA conjugate. As much as 55% of the stain waspresent as a diffuse high molecular weight band that resulted fromperiodate oxidation of the antibody.

EIA binding was determined for both the starting material and the finalproduct. EIA for the immunotoxin was 23.3% (relative to 100% forunmodified IND1) and the whole cell kill activity was >200 ng/mL for theAffi-gel Blue purified material.

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that variations andmodifications in terms of the molecular structures, preparationprocedures and reaction conditions may be made without departing fromthe spirit and scope of the invention.

What is claimed is:
 1. A compound having the formula ##STR7## in whichR¹ is a member selected from the group consisting of NH₂ -- and NH₂--NH--;R³ is a member selected from the group consisting of C₁ -C₁₀alkylene, phenyl-substituted C₁ -C₁₀ alkylene, benzyl-substituted C₁-C₁₀ alkylene, and amino-substituted C₁ -C₁₀ alkylene; and R⁴ is amember selected from the group consisting of H, acetyl, ##STR8## whereR⁵ is alkyl; and analogs selected from the group consisting ofwater-soluble salts and water-soluble derivatives thereof.
 2. A compoundhaving the formula ##STR9## in which R¹ is a member selected from thegroup consisting of NH₂ -- and NH₂ --NH--;R³ is a member selected fromthe group consisting of C₁ -C₁₀ alkylene, phenyl-substituted C₁ -C₁₀alkylene, benzyl-substituted C₁ -C₁₀ alkylene, and amino-substituted C₁-C₁₀ alkylene; and R⁴ is a member selected from the group consisting ofH, acetyl, ##STR10## where R⁵ is alkyl; and analogs selected from thegroup consisting of water-soluble salts and water-soluble derivativesthereof.
 3. A compound having the formula ##STR11## in which R¹ is amember selected from the group consisting of NH₂ -- and NH₂ --NH--;R³ isa member selected from the group consisting of C₁ -C₁₀ alkylene,phenyl-substituted C₁ -C₁₀ alkylene, benzyl-substituted C₁ -C₁₀alkylene, and amino-substituted C₁ -C₁₀ alkylene; and R⁴ is a memberselected from the group consisting of acetyl, 2-pyridylthio,4-pyridylthio, ##STR12## where R⁵ is alkyl; and analogs selected fromthe group consisting of water-soluble salts and water-solublederivatives thereof.
 4. A compound in accordance with claims 1, 2, or 3in which R³ is C₁ -C₁₀ alkylene.
 5. A compound in accordance with claims1, 2, or 3 in which R⁴ is a member selected from the group consisting ofH, acetyl and ##STR13##
 6. A compound in accordance with claims 1 or 2in which R³ is C₁ -C₁₀ alkylene, and R⁴ is a member selected from thegroup consisting of H, acetyl and ##STR14##
 7. A compound in accordancewith claims 1 or 2 in which R⁴ is a member selected from the groupconsisting of acetyl, 2-pyridylthio, and 4-pyridylthio.
 8. A compound inaccordance with claims 1, 2, or 3 in which said analogs arewater-soluble salts thereof selected from the group consisting ofacetate salts, trifluoroacetate salts, hydrohalide salts, andtoluenesulfonic acid salts.
 9. A compound in accordance with claims 1,or 2 in which R¹ is a meta- or para-position.
 10. A compound inaccordance with claims 1, or 2 in which R¹ is a at a para-position. 11.A compound having the formula ##STR15## in which R¹ is 4-amino, R³ is--(CH₂)₃ --, and R⁴ is acetyl.
 12. A compound having the formula##STR16## in which R¹ is 4-NH₃ ^(+-OOCCF) ₃, R³ is --(CH₂)₃ --, and R⁴is acetyl.
 13. A compound having the formula ##STR17## in which R¹ is4-amino, R³ is --(CH₂)₄ --, and R⁴ is acetyl.
 14. A compound having theformula ##STR18## in which R¹ is 4-NH₃ ^(+-OOCCF) ₃, R³ is --(CH₂)₄ --,and R⁴ is acetyl.
 15. A compound in accordance with claim 1 in which R¹is 4-amino, R³ is --CH₂ --, and R⁴ acetyl.
 16. A compound in accordancewith claim 1 in which R¹ is 4-NH₃ ^(+-OOCCF) ₃, R³ is --CH₂ --, and R⁴is acetyl.
 17. A compound in accordance with claim 1 in which R¹ is4-amino, R³ is --(CH₂)₂ --, and R⁴ is acetyl.
 18. A compound inaccordance with claim 1 in which R¹ is 4-NH₃ ^(+-OOCCF) ₃, R³ is--(CH₂)₂ --, and R⁴ is acetyl.
 19. A compound in accordance with claim 2in which R¹ is 4-amino, R³ is --(CH₂)₂ --, and R⁴ is 2-pyridylthio. 20.A compound in accordance with claim 2 in which R¹ is 4-NH₃ ^(+-OOCCF) ₃,R³ is --(CH₂)₂ --, and R⁴ is 2-pyridylthio.
 21. A compound in accordancewith claim 2 in which R¹ is 4-hydrazino, R³ is --(CH₂)₂ --, and R⁴ isacetyl.
 22. A compound in accordance with claim 2 in which R¹ is 4-NHNH₃^(+-OOCCF) ₃, R³ is --(CH₂)₂ --, and R⁴ is acetyl.
 23. A compound inaccordance with claim 2 in which R¹ is 4-hydrazino, R³ is --(CH₂)₂ --,and R⁴ is 2-pyridylthio.
 24. A compound in accordance with claim 2 inwhich R¹ is 4-NHNH₃ ^(+-OOCCF) ₃, R³ is --(CH₂)₂ --, and R⁴ is2-pyridylthio.
 25. A compound in accordance with claim 3 in which R¹ ishydrazino, R³ is --CH₂ --, and R⁴ is acetyl.
 26. A compound inaccordance with claim 3 in which R¹ is NHNH₃ ^(+-OOCCF) ₃, R³ is --CH₂--, and R⁴ is acetyl.
 27. A compound in accordance with claim 3 in whichR¹ is hydrazino, R³ is --(CH₂)₂ --, and R⁴ is 2-pyridylthio.
 28. Acompound in accordance with claim 3 in which R¹ is NHNH₃ ^(+-OOCCF) ₃,R³ is --(CH₂)₂ --, and R⁴ is 2-pyridylthio.
 29. A compound in accordancewith claim 1 in which R¹ is 4-amino, R³ is --(CH₂)₂ --, and R⁴ is2-pyridylthio.